Project Summary
Arthropod-borne flaviviruses and respiratory-transmitted coronaviruses have the potential to cause severe
epidemics and pandemics. One strategy to prepare for and respond to viral outbreaks is to develop drugs that
target host factors viruses require to complete their lifecycles. Through a series of CRISPR/Cas9 gene disruption
screens, we identified transmembrane protein 41B (TMEM41B) and the closely related vacuole membrane
protein 1 (VMP1) as critical pan-flavivirus and pan-coronavirus host factors. Both proteins are highly conserved
lipid scramblases with roles in autophagy. Our current model is that viruses from both the Flavivirdae and
Coronaviridae families hijack TMEM41B and VMP1 for their ability to remodel ER membranes and induce
membrane curvature to establish membrane-protected viral RNA replication organelles.
Our overall goal for this proposal is to understand how, on a mechanistic level, both proteins support
flavivirus and coronavirus infection. Our previous work indicates that TMEM41B is required at a post-entry step
at or prior to viral RNA replication. In Aim 1, we will interrogate early events of the virus lifecycle including primary
translation, polyprotein processing, and replication organelle formation in WT, TMEM41B and VMP1 knockout
(KO) cells to determine how far the flavivirus and coronavirus lifecycles progress in the absence of either protein.
We previously showed that lack of TMEM41B and VMP1, induces a heightened innate immune response
upon flavivirus infection. We hypothesize that both proteins are recruited to sites of viral RNA replication, and
that in their absence, RNA replication initiates and viral double stranded RNA (dsRNA) is produced. However,
without a proper replication organelle dsRNA is exposed and triggers an innate immune response. Alternatively,
given TMEM41B’s and VMP1’s lipid scramblase activity and function in lipid homeostasis, their absence may
induce ER stress, which triggers an unfolded protein response (UPR) that in synergy with dsRNA may cause a
heightened innate immune response. In Aim 2, we will test virus infection in double KO cells that lack either
protein in addition to genes that are essential for pathogen sensing, IFN signaling, and UPR activation. We will
further conduct RNAseq experiments to investigate lack of TMEM41B in stem cells and stem cell-derived
primary-like cells representing different tissue lineages in the absence and presence of viral replication.
Lastly, in Aim 3, will use a panel of phenotypic and mechanistic assays to characterize naturally occurring
SNPs in TMEM41B that we previously found to impact flavivirus replication, and several reported VMP1 loss-of-
function mutants. We will further take a deep mutational scanning approach to comprehensively characterize
TMEM41B and VMP1 and determine if any domains or amino acids are differentially required for their cellular
and proviral functions. This functional characterization will identify mutants that can be studied in detail in
mechanistic assays and may identify amino acids or interfaces in both proteins that can be targeted to prevent
virus infection with minimal disruption to cellular biology.